1. Field of the Invention
This invention relates to factor IX protein, a protein involved in the blood-clotting mechanism of warm-blooded animals, and its production by recombinant DNA technology.
2. Description of Prior Art
Haemophilia B, or Christmas disease, is an inherited, X-linked bleeding disorder caused by a defect in clotting factor IX. Injection of factor IX concentrate obtained from blood donors allows most patients to be successfully managed. However, due to impurities in the factor IX concentrate in use at present, this treatment involves some risk of infection by blood-borne viruses such as non-A, non-B hepatitis virus and the virus that causes AIDS. Despite recent apparent success in the heat-inactivation of the virus which causes AIDS, non-A non-B hepatitis virus remains resistant, see P. M. Mannucci et al., Lancet (ii) page 1013 (Nov. 2, 1985). Because of the considerable risk of viral infection in haemophiliacs, a factor IX preparation derived from a source other than blood is desirable.
Factor IX DNA was cloned in 1982, see K. H. Choo et al., Nature 299, 178-180 (1982), K. Kurachi et al., Proc. Nat. Acad. Sci. USA 79, 6461-6464 (1982) and European Patent Application Publication No. 107278A (NRDC). The work is summarised with sequence data and genome maps by D. S. Anson et al., EMBO J., 3 1053-1060 (1984) as well as in the above Patent Application.
Factor IX is a plasma glycoprotein which plays an essential role in the middle phase of the intrinsic clotting pathway where, in an activated form, IXa, it interacts with factor VIII(C), phospholipid and calcium ions to form a complex that converts factor X to Xa. Factor IX is synthesised in liver hepatocytes where the 461 amino acid long primary translation product (precursor) undergoes at least two stages of protein processing involving peptide cleavage as well as three distinct types of post-translational modification, before secretion into the bloodstream as a 415 amino acid long mature, biologically active glycoprotein.
The post-translational modifications are the vitamin K-dependent carboxylation of twelve glutamic acid residues, the addition of several carbohydrate residues and the beta-hydroxylation of a single aspartic acid residue. The first two modifications are known to be required for activity. Additionally, "prepeptide" and "propeptide" sequences totalling 46 amino acids have been removed as part of the processing of the precursor to give the mature, active protein. The precursor is not active in the blood-clotting pathway. Because of the complex and specialised nature of the processing and these modifications, it seemed probable that the expression of active factor IX derived from factor IX DNA clones, would present great problems.
The difficulties of obtaining a fully biologically active factor IX protein are amply illustrated by prior art which has been published between the priority dates and filing date of the present application.
L. Wasley et al., Blood, Supplement to the November 1985 issue, page 1256 describe expression of a factor IX gene in Chinese hamster ovary cells. The cells secreted over 100 micrograms/ml of a material having the antigenic characteristics of factor IX but of which only 1.5 micrograms/ml was native factor IX, i.e. was biologically active. The authors commented: "Current efforts are directed at improving the efficiency of gammacarboxylation and increasing the percentage of Factor IX that is biologically active."
Transgene S. A. filed a French Patent Application 8407125 on May 9, 1984 which has been published on Nov. 15, 1985 as U.S. Pat. No. 2,564,106. There are foreign counterparts, also published, including W085/05125 (Japan, USA) and European Patent Application Publication No. 167420A. These specifications describe cloning of the factor IX gene in E. coli and transfecting mammalian cells by theccalcium phosphate precipitation method. Mice embryo fibroblast cells were thus transfected, resulting in expression of proteins of molecular weight 62,000 and 72,000 daltons. Both proteins reacted with anti-factor IX antibodies, the 62,000 dalton protein was shown to be glycosylated, and a preliminary study showed that the antigen had carboxylated glutamic acid groups. The specifications speculated that the proteins thus obtained were factor IX precursors. That of molecular weight 62,000 daltons appears to be the primary translation product, since its extra molecular weight of about 5,000 daltons is readily accounted for by its possessing the additional 46 amino acids referred to above (assuming an average of molecular weight of 112 for an amino acid, 46.times.112=5152). It is noteworthy that Transgene S.A. did not perform a clotting assay or do any other test to indicate that the protein precursors described were biologically active. In all the circumstances, therefore, the reasonable conclusion is that the Transgene S.A. specifications do not describe mature, fully or even near-fully biologically active factor IX protein.
It is interesting that Transgene S.A. refer to the possibility of transfecting bovine kidney (MDBK) and monkey kidney (VERO) cell lines, see page 49 of the French specification of May 9, 1984 and in this connection say (in translation): "Some results have been obtained from the cells N1H-3T3 and LMTK (the mouse embryo fibroblasts) and are actually in progress for the lines MDBK and VERO". However, in the specifications filed at the end of the convention priority year in May 1985, precisely the same statement (word for word identical) appears. The reasonable conclusion is that there were considerable difficulties associated with the work in progress or that it was not felt worthwhile pursuing further the possibility of using these other cell lines.
Transgene S.A. also filed French patent applications on May 22, 1984 and Oct. 5, 1984, from which European Patent Application Publication No. 162782A published Nov. 27, 1985 claims priority. This specification describes expression of factor IX in vaccinia virus and cowpox virus. See also H. de la Salle et al., Nature 316, 268-270 (Jul. 18, 1985). The procedure involves infecting cells in which the poxvirus grows but which were ultimately killed by the virus. This gives high yields while the virus is growing, but which rapidly decrease to zero (usually within 24 hours). Control of the process presents serious problems for this reason and because there are two "biological reactants" requiring careful quality control, the cells and the virus stock. Substantial amounts of product failed to adsorb to barium sulphate, indicating that it had only about 50% of the biological activity of normal plasma factor IX. An even more serious problem is that the factor IX protein thus produced would have to be purified rigorously to remove live poxviral particles and antigens from the dead poxvirus, before the factor IX protein could be given to human patients. Vaccinia virus is a Class 2 human pathogen and can kill people who are seriously immunosuppressed.
Production of 50-60% biologically active human factor IX by transfection of Baby Hamster Kidney cells with factor IX cDNA has been reported by S. Busby et al., Nature 316, 271-273 (Jul. 18, 1985).
The subject matter of the present invention was published by D. S. Anson et al., Nature 315, 683-685 (Jun. 20, 1985).